Project Summary Rhabdomyosarcoma (RMS) is a devastating malignancy of muscle that is diagnosed in hundreds of children and adults annually in the USA. Survival rates are less than 50% in patients with unresectable, metastatic, or relapsed RMS. Thus, there is great clinical interest in elucidating key molecular pathways and genetic factors that are involved in continued RMS growth, tumor maintenance, and metastasis. The long-term goal and major clinical imperative of my work is to identify new actionable drug targets in RMS. My overall objective is to determine the extent to which PlexinA1 (PLXNA1) signaling regulates RMS growth, progression, and metastasis. My central hypothesis is that PLXNA1 is an important moderator of tumor growth in a large fraction of human RMS and likely regulates proliferation and metastasis through modulation of RHOA signaling. The rationale for prioritizing PLXNA1 for further studies comes from my preliminary data showing that PLXNA1 is expressed >8 fold in metastatic lesions found in zebrafish embryonal RMS, is transcriptionally upregulated in >95% of human RMS and is required for proliferation, anchorage dependent growth and migration of human RMS cells. Roles for Plexins in modulating RMS growth have not been fully defined. Plexins are a large family of transmembrane receptors that bind semaphorins to regulate intracellular signaling. Plexins regulate integrin functions and cytoskeletal dynamics through intrinsic GTPase activity and affect the recruitment of regulatory molecules including RHOA, modulating cell adhesion, migration, cell cycle progression and transformation. Our work has already implicated RHOA as a major regulator of proliferation and metastasis in human RMS, yet the upstream regulation by PLXNA1 has not been defined. Aim 1 will assess roles for PLXNA1 in tumor onset, growth, and metastasis in the zebrafish ERMS model, testing the hypothesis that PLXNA1 accelerates RMS onset, increases tumor growth and metastasis. Aim 2 will assess roles for PLXNA1 in RMS growth and maintenance using mouse xenografts, testing the hypothesis that PLXNA1 is required for xenograft RMS growth and maintenance. This work is important because it credentials the PLXNA1 signaling pathway as a therapeutic target in RMS. Aim 3 will identify the downstream molecular pathways regulated by the PLXNA1, testing my hypothesis that PLXNA1 activates RHOA to augment proliferation and metastasis in human RMS. With respect to outcomes, this work will 1) identify the role of PLXNA1 in RMS growth and metastasis, and 2) discover novel downstream targets of PLXNA1, likely providing new biomarkers for assessing drug effects on RMS and identifying additional drug targets for the treatment of this disease. Our work will likely have positive translational impact by providing novel mechanistic insights into how PLXNA1 functions in RMS and establish this pathway as a bona fide therapeutic target in human RMS. This work is clinically important because it lays the foundation for studying new PLXNA1 inhibitory compounds in RMS including PLXNA1 blocking antibodies and small molecule inhibitors to PLXNA1-NRP receptor activating SEMA3A ligands.